Potential deleterious effect of beta-adrenergic stimulation during warm-blood cardioplegia in rabbit hearts.

We hypothesized that beta-adrenergic stimulation with isoproterenol during continuous normothermic cardioplegic arrest would enhance the regenerative and regulatory function of the myocardium, resulting in improved cardiac function. We studied isolated rabbit hearts paced at approximately 200 beats per minute (bpm) and perfused by a support rabbit. We measured ventricular pressure over a range of ventricular volumes to determine maximal elastance (Emax) at baseline and 20 and 45 min after discontinuation of cardioplegia. Myocardial oxygen consumption (MVO2) measurements were performed simultaneously and during cardioplegic arrest. Hearts were prospectively randomized to receive either isoproterenol at 0.1 M or control in blinded fashion for 10 min during a 1-h continuous warm-blood cardioplegic arrest. Compared to control hearts, isoproterenol-treated hearts had trends toward longer time to first spontaneous heartbeat (control 141 +/- 43 vs. isoproterenol 200 +/- 74 s, p = .07), and longer time to capture of atrial pacing (control 214 +/- 52 vs. isoproterenol 288 +/- 91 s, p = .06). There was no difference observed in the MVO2 between isoproterenol-treated and control groups of hearts. MVO2 decreased during cardioplegia (p < .01), but there was no significant change in MVO2 during isoproterenol infusion during cardioplegic arrest. There was a significant reduction in Emax compared to baseline 20 min after discontinuation of cardioplegic arrest in both groups (control 7.3 +/- 1.7 mm Hg/microL vs. 9.0 +/- 1.7 mm Hg/microL, p = .02, isoproterenol-treated 6.8 +/- 2.8 mm Hg/microL vs. 8.2 +/- 2.6 mm Hg/microL, p = .01, respectively), with recovery of Emax by 45 min in control hearts only. We conclude that exposure of hearts to isoproterenol during warm cardioplegic arrest has a deleterious effect that may be mediated through mechanisms independent of increased myocardial oxygen consumption.

Leukocyte activation does not mediate myocardial leukocyte retention during endotoxemia in rabbits.

Our goal was to determine whether coronary leukocyte retention after endotoxin infusion was due primarily to leukocyte activation. Leukocytes were activated by infusion of endotoxin into 12 blood donor rabbits. Separately, 12 isolated rabbit hearts were perfused with blood from an endotoxemic support rabbit to expose coronary endothelium to an inflammatory stimulus. During an infusion of 20 ml of donor blood into the isolated heart, the coronary transit time of leukocytes was determined by deconvolution of multiple measurements of injectate and collected leukocyte concentrations. With no leukocyte activation or inflammatory stimulation of endothelium, leukocyte transit time was 9.2 +/- 3.5 s, and 11.6 +/- 4.1 x 10(6) leukocytes were retained in the coronary circulation. Leukocyte activation alone did not alter transit time (9.8 +/- 3.2 s) or retention (9.3 +/- 4.6 x 10(6) leukocytes). Inflammatory stimulation of endothelium with and without leukocyte activation increased transit time (18.0 +/- 3.6 and 18.9 +/- 3.8 s, respectively; P < 0. 05) and retention (24.8 +/- 8.4 and 25.3 +/- 6.8 x 10(6) leukocytes, respectively; P < 0.05) to the same extent. Differential counts showed that neutrophils (but not lymphocytes) were slowed and retained. Inflammatory stimulation of endothelium caused coronary capillary endothelial swelling and pseudopod formation. Thus increased coronary neutrophil transit time and retention are due to structural changes of coronary endothelial cells or other effects of the inflammatory response occurring within coronary capillaries, not only due to activation of leukocytes.

Effect of crystalloid administration on oxygen extraction in endotoxemic pigs.

We asked whether crystalloid administration improves tissue oxygen extraction in endotoxicosis. Four groups of anesthetized pigs (n = 8/group) received either normal saline infusion or no saline and either endotoxin or no endotoxin. We measured whole body (WB) and gut oxygen delivery and consumption during hemorrhage to determine the critical oxygen extraction ratio (ERO2 crit). Just after onset of ischemia (critical oxygen delivery rate), gut was removed for determination of area fraction of interstitial edema and capillary hematocrit. Radiolabeled microspheres were used to determine erythrocyte transit time for the gut. Endotoxin decreased WB ERO2 crit (0.82 +/- 0.06 to 0.55 +/- 0.08, P < 0.05) and gut ERO2 crit (0.77 +/- 0.07 to 0.52 +/- 0.06, P < 0.05). Unexpectedly, saline administration also decreased WB ERO2 crit (0.82 +/- 0.06 to 0.62 +/- 0.08, P < 0.05) and gut ERO2 crit (0.77 +/- 0.07 to 0.67 +/- 0.06, P < 0.05) in nonendotoxin pigs. Saline administration increased the area fraction of interstitial space (P < 0.05) and resulted in arterial hemodilution (P < 0.05) but not capillary hemodilution (P > 0.05). Saline increased the relative dispersion of erythrocyte transit times from 0.33 +/- 0.08 to 0.72 +/- 0.53 (P < 0.05). Thus saline administration impairs tissue oxygen extraction possibly by increasing interstitial edema or increasing heterogeneity of microvascular erythrocyte transit times.

L-2-Oxothiazolidine-4-carboxylic acid prevents endotoxin-induced cardiac dysfunction.

We tested the hypothesis that treatment with the glutathione repleting agent, L-2-oxothiazolidine-4-carboxylic acid (OTZ), could prevent endotoxin-induced ventricular dysfunction. Rabbits were treated with OTZ 2.4 g/kg (10% solution subcutaneously), or an equal volume and osmolality of saline, 24 h prior to, and again (intravenously) just prior to, infusion of 1 mg/kg E. coli endotoxin (or vehicle control). Ventricular contractility was measured in isolated hearts perfused by support rabbits. Contractility did not change in control groups (Saline/Control [n = 7] or OTZ/Control [n = 7]) over 6 h. However, Emax decreased in the Saline/Endotoxin group (-16.1 +/- 4.5% from baseline, n = 7, p < 0.05) and this was prevented by pretreatment with OTZ in the OTZ/ Endotoxin group (+6.3 +/- 4.1%, n = 7, p < 0.05 by analysis of variance). To better understand the mechanism of this effect we measured myocardial glutathione concentration and found it to be greater in OTZ/Endotoxin animals (104 +/- 4 ng/g) than in the Saline/Endotoxin animals (80 +/- 3 ng/g, p < 0.05). OTZ did not appreciably alter the endotoxin-induced increase in serum concentration of tumor necrosis factor (TNF) or the endotoxin-induced increase in myocardial leukocyte content. We conclude that oxygen radicals contribute to the early decrease in left ventricular contractility after endotoxin infusion and this decrease may be prevented by OTZ.

Leukocytes and decreased left-ventricular contractility during endotoxemia in rabbits.

We hypothesized that leukocytes contribute to decreased myocardial contractility following endotoxin infusion. To test this hypothesis, we administered endotoxin (1 mg/kg intravenously) to intact, anesthetized rabbits whose arterial blood perfused two isolated hearts at a constant pressure (75 mm Hg). One heart was perfused with blood passed through a leukocyte filter, whereas the other received unfiltered blood. Contractility of both hearts was measured every 30 min for 6 h, using the slope of the end-systolic pressure-volume relationship (Emax) and the maximum rate of change of intraventricular pressure (dP/dt(max)). In the unfiltered hearts at 6 h, Emax decreased to 81 +/- 6% (mean +/- SEM) of baseline (p < 0.05). In the hearts perfused with leukocyte-filtered blood there was no change in Emax. Similarly, dP/dt(max) decreased 74 +/- 9% of baseline in the hearts receiving unfiltered blood (p < 0.05) but did not decrease in the hearts receiving leukocyte-filtered blood. The leukocyte filter significantly reduced the number of neutrophils in perfusing blood (p < 0.01), decreased the number of neutrophils in the heart by 77% (p < 0.01), and decreased myocardial morphometric changes (p < 0.05). A 55 +/- 18% reduction in neutrophil L-selectin expression after endotoxin infusion (p < 0.01) suggests that the neutrophils were significantly activated. We conclude that leukocytes, notably activated neutrophils, may contribute to decreased myocardial contractility during septic shock.

The Drosophila Eip78C gene is not vital but has a role in regulating chromosome puffs.

We have generated a number of chromosomal aberrations that disrupt the early-late ecdysone-induced 78C puff gene (Eip78C, ecdysone-induced protein, FlyBase name for the E78 gene of Stone and Thummel 1993), which encodes the two members of the nuclear hormone receptor superfamily Eip78C-A and Eip78C-B. The aberrations include deletions of the ligand-binding/dimerization domain of both, inversions that split Eip78C-A but retain residual Eip78C-B expression, and a small deletion specific for Eip78C-B. We find that wild-type Eip78C functions are completely dispensable for normal development under laboratory conditions. However, we show that Eip78C-B is required for the maximal puffing activity of a subset of late puffs (63E and 82F) since these puffs are reduced in size in Eip78C-B mutant backgrounds. Paradoxically the same late puffs are reduced, as well as at least one other, when the Eip78C-B cDNA is overexpressed from a heat shock promoter. These data indicate either that Eip78C function is redundant or that it plays a subtle modulating role in the regulation of chromosome puffing.

Heterogeneity of gut capillary transit times and impaired gut oxygen extraction in endotoxemic pigs.

We tested the hypothesis that endotoxin increases the heterogeneity of gut capillary transit times and impairs oxygen extraction. The gut critical oxygen extraction ratio was determined by measuring multiple oxygen delivery-consumption points during progressive phlebotomy in eight control and eight endotoxin-infused anesthetized pigs. In multiple 1- to 2-g samples of small bowel, we measured blood volume (radiolabeled red blood cells) and flow (radiolabeled 15-microns microspheres) before and after critical oxygen extraction. Red blood cell transit time (= volume/flow) multiplied by morphologically determined capillary/total blood volume gave capillary transit time. During hemorrhage, capillary/total blood volume did not change in the endotoxin group (0.5 +/- 4.5%) but increased in the control group (17.6 +/- 2.5%; P < 0.05) due to a decrease in total gut blood volume. Flow decreased significantly in the endotoxin group (36 +/- 10%; P < 0.05) but not in the control group (12 +/- 10%). Capillary transit-time heterogeneity increased in the endotoxin group (12.3 +/- 4.9%) compared with the control group (-5.8 +/- 7.4%; P < 0.05), predicting a critical oxygen extraction ratio 0.14 lower in the endotoxin group than in the control group (K. R. Walley. J. Appl. Physiol. 81: 885-894, 1996). This matches the measured difference (endotoxin group, 0.60 +/- 0.04; control group, 0.74 +/- 0.03; P < 0.05). Increased heterogeneity of capillary transit times may be an important cause of impaired oxygen extraction.

Myocardial morphometric changes related to decreased contractility after endotoxin.

Decreased ventricular contractility during sepsis lasts much longer than the half-lives of inflammatory mediators that have been suggested to be myocardial depressant factors. Our hypothesis is that blood-borne factors may also cause myocardial structural changes, including damage and death of myocytes, associated with decreased ventricular contractility. We tested this hypothesis in an isolated rabbit heart perfused by a support rabbit. Support rabbits received 1 mg/kg endotoxin i.v. over 30 min (endotoxin group, n = 7) or vehicle (control group, n = 6). The slope of the end-systolic pressure-volume relationship, Emax, was used to measure contractility of the isolated heart. Five hours after endotoxin infusion, Emax decreased by 17 +/- 7% (P < 0.03) compared with 0 +/- 2% in the control group. Quantitative morphometric analysis of isolated hearts from the endotoxin group demonstrated an increased volume fraction of myocardial capillaries occupied by leukocytes (15.7 +/- 3.5 vs. 3.0 +/- 0.7% in the control group, P < 0.05), structurally abnormal myocytes (7.6 +/- 3.6 vs. 0.8 +/- 0.4%, P < 0.05), and interstitial edema (23.2 +/- 5.2 vs. 14.3 +/- 2.1%, P < 0.05). We conclude that blood-borne factors cause myocardial structural changes that may contribute to decreased ventricular contractility and may explain the prolonged decrease in ventricular contractility during sepsis.

Prolonged leukocyte transit time in coronary microcirculation of endotoxemic pigs.

We quantified the timing and extent of leukocyte retention by the coronary microcirculation in a pig model of hyperdynamic sepsis in three ways. First, the transmyocardial leukocyte gradient was determined as coronary blood flow (calibrated ultrasonic flow probe) multiplied by the difference between leukocyte counts in the aorta and coronary sinus. Measurements were taken at 1-min intervals for 30 min and then at 3-min intervals for 45 min in anesthetized pigs exposed to either endotoxin (50 micrograms/kg iv over 30 min) (n = 7) or vehicle (n = 7). Second, postmortem morphometric analysis was used to quantitate the number and location of retained myocardial leukocytes. Finally, myocardial capillary transit time of leukocytes was calculated from the above measures. In the endotoxin group 2.1 +/- 0.8 x 10(9) leukocytes/100 g wet wt were retained in the coronary circulation, primarily in capillaries. This resulted in 111 +/- 37 (P < 0.05) times as many leukocytes in the coronary microcirculation than predicted from the arterial leukocyte concentration. Myocardial capillary transit time of leukocytes was prolonged to 39.1 +/- 20.6 s (P < 0.05) in the endotoxin group versus 5.0 +/- 1.4 s in the control group. We conclude that, after endotoxin infusion in a pig model of hyperdynamic sepsis, myocardial leukocyte transit is slowed, leading to the retention of large numbers of leukocytes in the coronary microcirculation.

Anti-tumor necrosis factor-alpha prevents decreased ventricular contractility in endotoxemic pigs.

It is not known how the decrease in left ventricular contractility following endotoxin exposure is mediated, or whether this decrease is preventable by antibodies to tumor necrosis factor-alpha (TNF alpha). Four groups of six anesthetized and instrumented pigs were pretreated with ovine polyclonal antibody to human TNF alpha (anti-TNF alpha), nonspecific IgG, or saline, and then treated with either endotoxin or saline. We measured hemodynamics and left ventricular pressures (Millar catheter) and volumes (conductance catheter). Left ventricular contractility was assessed using the slope (Emax) of the end-systolic pressure-volume relationship. Four hours after the start of endotoxin infusion in the nonspecific IgG pretreated group, Emax had decreased by 44 +/- 6% (p < 0.05), mean arterial pressure had decreased from 115 +/- 7 mm Hg to 70 +/- 10 mm Hg (p < 0.05), and cardiac output was rapidly decreasing after an initial increase (p < 0.05). Anti-TNF alpha significantly reduced the decrease in Emax (11 +/- 9%, p < 0.05), and the systemic hypotension (108 +/- 15 mm Hg to 99 +/- 6 mm Hg, p < 0.05), at 4 h, and prevented the late decrease in cardiac output. This suggests that TNF alpha is an important early mediator in sepsis leading to decreased left ventricular contractility.

Birthdates of trigeminal ganglion cells contributing axons to the infraorbital nerve and specific vibrissal follicles in the rat.

Prenatal labelling with [3H]-thymidine was combined with retrograde tracing techniques in adult rats to determine the birthdates of the trigeminal (V) ganglion cells that contributed axons to the infraorbital nerve (ION) and the generation of the subsets of ION cells that innervated specific vibrissae follicles (C-1 and C-5). The V ganglion cells contributing axons to the ION are born between embryonic (E-, E-0 = the day of conception) days 9.5 and 14.5. The percentages (normalized so that they total 100%) of the total V ganglion population born on E-9.5 through E-14.5 were 5.8, 25.7, 19.8, 23.4, 21.0, and 4.4%, respectively. The distribution of birthdates for the V ganglion cells that were retrogradely labelled from the ION closely matched that for the ganglion as a whole. All of these neurons were also born on E-9.5 through E-14.5, and the percentages born on each day were 6.3, 23.6, 18.1, 24.0, 23.6, and 4.4%. Finally, a similar distribution of birthdates was obtained for the V ganglion cells that were retrogradely labelled after injection of retrograde tracers into either the C-1 or C-5 vibrissae follicles. We were unable to detect any distinctive spatial distributions for either all V ganglion or ION cells born on a specific embryonic day. Furthermore, neurons with a given birthdate and that innervated a given follicle were distributed throughout the entire region containing all of the ganglion cells supplying the follicle in question. Therefore, it appears that the V ganglion cells contributing axons to the ION are born over the entire period of ganglion neurogenesis and further that the organization of the ION's innervation of the periphery is not a function of cell birthdate.